Evaluating Fossil Calibrations for Dating Phylogenies in Light of Rates of Molecular Evolution: a Comparison of Three Approaches
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Syst. Biol. 61(1):22–43, 2012 c The Author(s) 2011. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: [email protected] DOI:10.1093/sysbio/syr075 Advance Access publication on August 11, 2011 Evaluating Fossil Calibrations for Dating Phylogenies in Light of Rates of Molecular Evolution: A Comparison of Three Approaches 1,2, 3 1 VIMOKSALEHI LUKOSCHEK ∗, J. SCOTT KEOGH , AND JOHN C.AVISE 1Department of Ecology and Evolutionary Biology, University of California at Irvine, Irvine, CA 92697, USA; 2Australian Research Council Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, QLD 4811, Australia; and 3Division of Evolution, Ecology and Genetics, Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia; ∗Correspondence to be sent to: ARC Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, QLD 4811, Australia; E-mail: [email protected]. Received 17 November 2010; revises returned 21 January 2011; accepted 4 March 2011 Associate Editor: Frank E. Anderson Abstract.—Evolutionary and biogeographic studies increasingly rely on calibrated molecular clocks to date key events. Although there has been significant recent progress in development of the techniques used for molecular dating, many issues remain. In particular, controversies abound over the appropriate use and placement of fossils for calibrating molec- ular clocks. Several methods have been proposed for evaluating candidate fossils; however, few studies have compared the results obtained by different approaches. Moreover, no previous study has incorporated the effects of nucleotide sat- uration from different data types in the evaluation of candidate fossils. In order to address these issues, we compared three approaches for evaluating fossil calibrations: the single-fossil cross-validation method of Near, Meylan, and Shaffer (2005. Assessing concordance of fossil calibration points in molecular clock studies: an example using turtles. Am. Nat. 165:137–146), the empirical fossil coverage method of Marshall (2008. A simple method for bracketing absolute divergence times on molecular phylogenies using multiple fossil calibration points. Am. Nat. 171:726–742), and the Bayesian multical- ibration method of Sanders and Lee (2007. Evaluating molecular clock calibrations using Bayesian analyses with soft and hard bounds. Biol. Lett. 3:275–279) and explicitly incorporate the effects of data type (nuclear vs. mitochondrial DNA) for identifying the most reliable or congruent fossil calibrations. We used advanced (Caenophidian) snakes as a case study; however, our results are applicable to any taxonomic group with multiple candidate fossils, provided appropriate taxon sampling and sufficient molecular sequence data are available. We found that data type strongly influenced whichfos- sil calibrations were identified as outliers, regardless of which method was used. Despite the use of complex partitioned models of sequence evolution and multiple calibrations throughout the tree, saturation severely compressed basal branch lengths obtained from mitochondrial DNA compared with nuclear DNA. The effects of mitochondrial saturation were not ameliorated by analyzing a combined nuclear and mitochondrial data set. Although removing the third codon positions from the mitochondrial coding regions did not ameliorate saturation effects in the single-fossil cross-validations, it did in the Bayesian multicalibration analyses. Saturation significantly influenced the fossils that were selected as most reliable for all three methods evaluated. Our findings highlight the need to critically evaluate the fossils selected by data with different rates of nucleotide substitution and how data with different evolutionary rates affect the results of each method for evalu- ating fossils. Our empirical evaluation demonstrates that the advantages of using multiple independent fossil calibrations significantly outweigh any disadvantages. [Bayesian dating; Caenophidia; cross-validation; fossil calibrations; Hydrophi- inae; molecular clock; nucleotide saturation; snakes.] Ideally, molecular clock calibrations are obtained from existed than the clade’s common ancestor. Fossils also accurately dated fossils that can be assigned to nodes may be incorrectly assigned to the crown rather than with high phylogenetic precision (Graur and Martin the stem of a clade (Doyle and Donoghue 1993; Maga- 2004), but reality is generally far from this ideal be- llon and Sanderson 2001). The most useful fossils are, cause of a number of important problems. The incom- therefore, geologically well dated, preserved with suffi- plete and imperfect nature of the fossil record means cient morphological characters to be accurately placed that fossils necessarily only provide evidence for the on a phylogenetic tree and temporally close to an ex- minimum age of a clade. Many clades will be consider- tant node rather than buried within a stem lineage (van ably older than the oldest known fossil; thus, nodes may Tuinen and Dyke 2004). However, the fossil records of be constrained to erroneously young ages (Benton and many, if not most, taxonomic groups fall far short of Ayala 2003; Donoghue and Benton 2007; Marshall 2008). these criteria. As such, several methods have been de- Incorrect fossil dates also arise from experimental errors veloped for evaluating candidate fossil calibrations in in radiometric dating of fossil-bearing rocks or incor- order to: determine their internal consistency and iden- rectly assigning fossils to a specific stratum. In addition, tify outliers (Near et al. 2005), identify lineages with misinterpreted character state changes can result in the the best fossil coverage and identify outliers (Marshall taxonomic misidentification of fossils or their incorrect 2008), and evaluate alternative placements of fossils placement on the phylogeny (Lee 1999). Ideally, a fossil (Rutschmann et al. 2007; Sanders and Lee 2007). would date the divergence of two descendant lineages However, fossil calibrations are not the only difficulty from a common ancestor. In reality, however, fossils in molecular dating. Other factors also contribute to rarely represent specific nodes but rather points along a inaccurately calibrated molecular clocks including: in- branch (Lee 1999; Conroy and van Tuinen 2003). Thus, correctly specified models of evolution (Brandley et al. although a fossil may appear to be ancestral to a clade, 2011), inappropriate modelling of rate heterogeneity it is impossible to determine how much earlier the fossil among lineages (Sanderson 1997; Drummond et al. 22 2012 LUKOSCHEK ET AL.—EVALUATING FOSSIL CALIBRATIONS 23 2006), and unbalanced taxon sampling potentially re- evaluating fossil calibrations: the single-fossil cross- sulting in node-density artifacts (Hugall and Lee 2007). validation method of Near et al. (2005), the empirical In addition, choice of genetic data or gene region can fossil coverage method of Marshall (2008), and the strongly affect estimated divergences (Benton and Ayala Bayesian multicalibration method of Sanders and Lee 2003). For example, in rapidly evolving genes, such as (2007) and explicitly evaluate the effects of nucleotide mitochondrial DNA, saturation has been shown to have saturation on the results of each method. Briefly, the the effect of compressing basal branches and artificially single-fossil cross-validation approach (Near et al. 2005) pushing shallow nodes toward basal nodes, resulting in evaluates candidate fossils, including the alternative overestimated divergence dates (Hugall and Lee 2004; ages or placements of fossils at some calibrated nodes, Townsend et al. 2004; Hugall et al. 2007; Phillips 2009). with the aim of identifying a number of plausible reli- However, the nature of the bias is complicated. For able calibration sets. The approach of Marshall (2008) example, underestimating the true rate of hidden sub- aims to identify candidate calibrations with the best stitution results in tree compression: However, if the fossil coverage and then tests whether these fossils are rate of hidden substitutions were to be overestimated, potential outliers. Finally, the Bayesian multicalibration the reverse would be true. These effects are further approach evaluates one or more alternative calibrations complicated by the calibration placement. For exam- in a set by comparing the Bayesian prior and posterior ple, if only deep splits are calibrated, then recent nodes probabilities (PPs) at fossil-calibrated nodes (Sanders will be biased to be younger under tree extension and and Lee 2007). We explicitly evaluate the effects of using older under tree compression. Slowly evolving genes, sequence data with different rates of molecular evo- as are typical for nuclear DNA, are less prone to such lution on the best fossils identified by each method saturation effects; however, nuclear DNA data are not using the same mitochondrial and nuclear sequence completely immune to these issues; problems of satu- data set (each with identical taxon sampling) for each ration also can emerge for slowly evolving nuclear loci method. In addition, we evaluate whether saturation ef- if deeper divergences are being investigated. More im- fects can be ameliorated by 1) removing the third codon portantly, although the effects of saturation have been position of the mitochondrial coding regions and 2) an-